International Journal of Molecular Sciences Article Mycobacterial Epoxide Hydrolase EphD Is Inhibited by Urea and Thiourea Derivatives Jan Madacki 1, Martin Kopál 1, Mary Jackson 2 and Jana Korduláková 1,* 1 Department of Biochemistry, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská Dolina, Ilkoviˇcova6, 842 15 Bratislava, Slovakia; [email protected] (J.M.); [email protected] (M.K.) 2 Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA; [email protected] * Correspondence: [email protected] Abstract: The genome of the human intracellular pathogen Mycobacterium tuberculosis encodes an unusually large number of epoxide hydrolases, which are thought to be involved in lipid metabolism and detoxification reactions needed to endure the hostile environment of host macrophages. These enzymes therefore represent suitable targets for compounds such as urea derivatives, which are known inhibitors of soluble epoxide hydrolases. In this work, we studied in vitro the effect of the thiourea drug isoxyl on six epoxide hydrolases of M. tuberculosis using a fatty acid substrate. We show that one of the proteins inhibited by isoxyl is EphD, an enzyme involved in the metabolism of mycolic acids, key components of the mycobacterial cell wall. By analyzing mycolic acid profiles, we demonstrate the inhibition of EphD epoxide hydrolase activity by isoxyl and two other urea-based inhibitors, thiacetazone and AU1235, inside the mycobacterial cell. Keywords: mycobacterium; epoxide hydrolase; isoxyl; thiacetazone; AU1235; mycolic acids Citation: Madacki, J.; Kopál, M.; Jackson, M.; Korduláková, J. Mycobacterial Epoxide Hydrolase EphD Is Inhibited by Urea and 1. Introduction Thiourea Derivatives. Int. J. Mol. Sci. With about 10 million new cases and 1.4 million deaths in 2019, tuberculosis (TB), 2021, 22, 2884. https://doi.org/ a disease caused by pathogenic bacterium Mycobacterium tuberculosis, remains a serious 10.3390/ijms22062884 global health problem. The End TB Strategy adopted by the World Health Organization (WHO) envisioned a 20% decline in global incidence of TB and a 35% reduction in TB Academic Editor: Saša Frank deaths between 2015 and 2020; however, despite some progress, these milestones have not been reached [1]. Accelerating this decline and putting a stop to the TB epidemic Received: 29 January 2021 requires intensifying the search for a new vaccine and new chemotherapeutics, tasks which Accepted: 10 March 2021 face several challenges [2–4]. Currently, a six-month regimen is routinely implemented in Published: 12 March 2021 treatment of drug susceptible TB, consisting of a combination of isoniazid (INH), rifampicin (RIF), ethambutol (EMB) and pyrazinamide (PZA). In the case of multiple-drug-resistant Publisher’s Note: MDPI stays neutral TB (MDR-TB), the treatment can last up to 20 months and involves administration of with regard to jurisdictional claims in second-line TB drugs, which are associated with more severe side effects and toxicity [5]. published maps and institutional affil- Isoxyl (thiocarlide; 4,40-diisoamyloxydiphenylthiourea) (ISO) (Figure1) is a thiourea iations. compound that was used for treatment of TB in the 1960s, but its use was discontinued due to poor bioavailability [6]. Nevertheless, ISO shows potent antimycobacterial activity in vitro with low minimal inhibitory concentrations (MICs) on M. tuberculosis, including strains resistant to RIF and INH [7]. Initial studies found that ISO inhibits the stearoyl-CoA Copyright: © 2021 by the authors. D9-desaturase DesA3. However, this did not explain the inhibition of mycolic acids seen Licensee MDPI, Basel, Switzerland. in ISO treated M. tuberculosis [8]. Mycolic acids are very long chain (C60–C90) α-alkyl This article is an open access article β-hydroxy fatty acids and are key components of the mycobacterial outer membrane distributed under the terms and (mycomembrane), their presence being essential for the viability of the bacterium [9,10]. conditions of the Creative Commons Attribution (CC BY) license (https:// Mycobacteria produce several types of mycolates differing in the presence of different creativecommons.org/licenses/by/ functional groups in their hydrocarbon chain, such as double bonds, cyclopropane rings 4.0/). Int. J. Mol. Sci. 2021, 22, 2884. https://doi.org/10.3390/ijms22062884 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 11 Int. J. Mol. Sci. 2021, 22, 2884 comembrane), their presence being essential for the viability of the bacterium [9,10].2 My- of 10 cobacteria produce several types of mycolates differing in the presence of different func- tional groups in their hydrocarbon chain, such as double bonds, cyclopropane rings and oxygenatedand oxygenated functions functions [11]. In [11 subs]. Inequent subsequent studies, studies, it was shown it was that shown ISO and that thiacetazone ISO and thi- (TAC),acetazone a related (TAC), thiosemicarbazone a related thiosemicarbazone drug (Figure drug 1), both (Figure target1), bothHadAB, target the HadAB, β-hydroxy- the acylβ-hydroxyacyl-ACP-ACP dehydratase dehydratase component componentof the fatty of acid the synthase fatty acid II synthase (FASII) system, II (FASII) which system, is responsiblewhich is responsible for the synthesis for the synthesisof mycolic of acids mycolic [12]. acids It is well [12]. established It is well established that ISO and that TAC ISO areand prodrugs TAC are and prodrugs require and modification require modification by the mycobacterial by the mycobacterial monooxygenase monooxygenase EthA for ac- tivityEthA [13 for– activity15]. [13–15]. Figure 1. Figure 1. ChemicalChemical structures structures of of isoxyl isoxyl (ISO), (ISO), thiacetazone thiacetazone (TAC) (TAC) and AU1235. Mycobacteria have an extremely hydrophobic cell envelope that consists of a conven- Mycobacteria have an extremely hydrophobic cell envelope that consists of a conven- tional plasma membrane; a layer of peptidoglycan; and a polysaccharide, arabinogalactan, tional plasma membrane; a layer of peptidoglycan; and a polysaccharide, arabinogalactan, which is esterified by mycolic acids, forming the inner leaflet of the mycomembrane [16]. which is esterified by mycolic acids, forming the inner leaflet of the mycomembrane [16]. The outer leaflet of the mycomembrane is composed of various complex lipids such as The outer leaflet of the mycomembrane is composed of various complex lipids such as trehalose dimycolate (TDM), di- and polyacyl trehaloses, phthiocerol dimycocerosates and trehalosesulfolipids dimycolate [17,18]. The (TDM), mycobacterial di- and cellpolyacyl envelope trehaloses, is an important phthiocerol target dimycocerosates for antimycobac- andterial sulfolipids compounds, [17,18]. with The the mycobacterial most notable examplescell envelope being is an first-line important antituberculotics target for antimy- INH, cobacterialwhich inhibits compounds, the FASII enoyl-ACPwith the most reductase notable InhA examples [19,20 ],being and EMB,first-line which antituberculotics interferes with INHthe synthesis, which inhibits of arabinogalactan the FASII enoyl [21].-ACP In recent reductase years, InhA several [19,20], new inhibitors and EMB were, which found inter- to ferestarget with the cellthe envelope,synthesis forof arabinogalactan example, by affecting [21]. theIn recent transporter years, MmpL3, several whichnew inhibitors transfers weretrehalose found monomycolate to target the cell (TMM) envelope, across thefor example plasma membrane, by affecting [22 ].the Once transporter transferred MmpL3, to the whichperiplasmic transfers space, trehalose TMM ismonomycolate a substrate for (TMM) the mycolyltransferases across the plasma FbpA,membrane FbpB [22]. and FbpCOnce transferred(also known to collectively the periplasmic as the antigenspace, TMM 85 complex) is a substrate that catalyze for the the myco transferlyltransferases of mycolic FbpA,acyl chains FbpB ontoand cellFbpC wall (also arabinogalactan known collectively or the as synthesis the antigen of trehalose 85 complex) dimycolate that catalyze (TDM), thethus transfer incorporating of mycolic mycolates acyl chains into theonto mycomembrane cell wall arabinogalactan [23]. One of or the the MmpL3 synthesis inhibitors of tre- haloseis an adamantyl dimycolate urea (TDM), AU1235 thus (Figure incorporating1), which mycolates inhibits a rangeinto the of mycobacterialmycomembrane strains, [23]. Oneincluding of the MDRMmpL3 isolates inhibitors of M. tuberculosisis an adamantyl[22]. urea AU1235 (Figure 1), which inhibits a rangeInitial of mycobacterial analysis of strains, the genome including of virulent MDR isolates strain M.of M. tuberculosis tuberculosisH37Rv [22]. discovered fiveInitial genes analysis encoding of putative the genome epoxide of virulent hydrolases strain (EHs)M. tuberculosis homologous H37Rv to eukaryoticdiscovered αfive/β geneshydrolase encoding fold EHs,putative each epoxide about 300hydrolases amino acids(EHs) long homologous (ephA, ephB, to eukaryotic ephC, ephE α/βand hydro-ephF), laseand fold another EHs, one each which about appeared 300 amino to be acids an N-terminal long (ephA, part ephB, of a fusionephC, ephE protein and with ephF a), short- and anotherchain dehydrogenase one which appeared (SDR) atto its be C-terminus, an N-terminal named partephD of a [fusion24,25]. protein Another with EH a from shortM.- chaintuberculosis dehydrogenase(EphG)